Plant hormone jasmonate prioritizes defense overgrowth by interfering with gibberellin signaling cascadeDong-Lei Yanga,1, Jian Yaob,c,1, Chuan-Sheng Meid,1,2, Xiao-Hong Tonga, Long-Jun Zenga, Qun Lia, Lang-Tao Xiaoe,Tai-ping Sunf, Jigang Lig, Xing-Wang Dengg, Chin Mei Leeb, Michael F. Thomashowb, Yinong Yangd,h,3, Zuhua Hea,3,and Sheng Yang Heb,c,3

aNational Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academyof Sciences, Shanghai 200032, China; bDepartment of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824; cHoward HughesMedical Institute, Chevy Chase, MD 20815; dDepartment of Plant Pathology, University of Arkansas, Fayetteville, AR 72701; eHunan Provincial Key Laboratoryof Phytohormones, Hunan Agricultural University, Changsha 410128, China; fDepartment of Biology, Duke University, Durham, NC 27705; gDepartment ofMolecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; and hDepartment of Plant Pathology and Huck Institutes of LifeSciences, Pennsylvania State University, University Park, PA 16802

Edited by Frederick M. Ausubel, Harvard Medical School, Massachusetts General Hospital, Boston, MA, and approved March 20, 2012 (received for reviewJanuary 30, 2012)

Plants must effectively defend against biotic and abiotic stressesto survive in nature. However, this defense is costly and is oftenaccompanied by significant growth inhibition. How plants co-ordinate the fluctuating growth-defense dynamics is not wellunderstood and remains a fundamental question. Jasmonate (JA)and gibberellic acid (GA) are important plant hormones thatmediate defense and growth, respectively. Binding of bioactiveJA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (theJAZ or DELLA family of proteins), which, at the resting state,represses cognate transcription factors involved in defense (e.g.,MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. Inthis study, we found that the coi1 JA receptor mutants of rice (adomesticated monocot crop) and Arabidopsis (a model dicot plant)both exhibit hallmark phenotypes of GA-hypersensitive mutants.JA delays GA-mediated DELLA protein degradation, and the dellamutant is less sensitive to JA for growth inhibition. Overexpres-sion of a selected group of JAZ repressors in Arabidopsis plantspartially phenocopies GA-associated phenotypes of the coi1 mu-tant, and JAZ9 inhibits RGA (a DELLA protein) interaction withtranscription factor PIF3. Importantly, the pif quadruple (pifq) mu-tant no longer responds to JA-induced growth inhibition, andoverexpression of PIF3 could partially overcome JA-inducedgrowth inhibition. Thus, a molecular cascade involving the COI1–JAZ–DELLA–PIF signaling module, by which angiosperm plants pri-oritize JA-mediated defense over growth, has been elucidated.

disease resistance | plant growth | plant immunity | light response |plant defense

Sessile plants have evolved a dynamic regulatory network toadapt to the daily and seasonally fluctuating environment.

Jasmonate (JA) is a lipid-derived plant hormone that regulatesdevelopmental processes, including pollen development, tendrilcoiling, fruit ripening and senescence, as well as response tobiotic and abiotic stress (1–3). The F-box protein coronatineinsensitive 1 (COI1), a component of the SCF E3 ubiquitin li-gase, has been identified as a principal component of a receptorof JA in Arabidopsis and other plants (4–7), and the JA ZIM-domain (JAZ) family proteins are key regulators of JA signalingthat repress transcription of JA-responsive genes through in-teraction with transcription factors, such as MYC2 (8–10). Thistranscriptional repression requires novel interactor of JAZ(NINJA) and TOPLESS corepressor proteins (11). BioactiveJA, the jasmonoyl-isoleucine conjugate, promotes physical in-teraction between COI1 and JAZ proteins that results in deg-radation of JAZs by the 26S proteasome, leading to initiation ofJA responses (9, 10). Therefore, the SCFCOI1

–JAZ proteincomplex acts as a core site of JA perception. As a regulatoryloop, JA also activates JAZ gene transcription, leading to the

down-regulation of JA action and the dynamic nature of JAresponse (12).Gibberellic acids (GAs) are plant growth-promoting hormones

that play important roles in diverse aspects of plant growth anddevelopment, such as stem elongation, leaf expansion, flowering,seed development, and germination (13–17). The DELLA fam-ily proteins are key regulators of GA signaling that represstranscription of GA-responsive genes through interaction withgrowth-promoting transcription factors, such as phytochromeinteracting factors (PIFs) (18, 19). Bioactive GAs bind to the GAinsensitive dwarf1 (GID1) receptor, which, in turn, interacts withDELLA proteins (five members in Arabidopsis and a single SLR1protein in rice) (16, 20). The GA-GID1–bound DELLA/SLR1proteins are recognized by an F-box protein (SLY1 in Arabi-dopsis and GID2 in rice), resulting in proteasome-dependentdegradation of DELLA/SLR1 repressors. Degradation ofDELLA/SLR1 proteins derepresses the transcriptional activitiesof downstream transcription factors, including PIFs (16).Activation of JA defense signaling is known to severely restrict

plant growth, representing a prominent example of growth–de-fense tradeoff in plants. There have been several reports ofcrosstalk between JA and GA signaling pathways in Arabidopsis,mostly documenting the antagonistic effect of GA on JA sig-naling (1). A quadruple della mutant (which lacks four of the fiveArabidopsis DELLA proteins) was shown to be partially in-sensitive to gene induction by JA, whereas the constitutivelyactive dominant DELLA mutant gai was found to be sensitizedfor JA-responsive gene induction, implicating DELLAs in JAsignaling and/or perception (21). Cheng et al. (22) found thatGA promotes JA biosynthesis, thereby inducing the expressionof MYB21, MYB24, and MYB57 to promote stamen filament

Author contributions: D.-L.Y., J.Y., C.-S.M., Y.Y., Z.H., and S.Y.H. designed research; D.-L.Y.,J.Y., C.-S.M., X.-H.T., L.-J.Z., L.-T.X., Y.Y., and Z.H. performed research; C.-S.M., Q.L., T.-p.S.,J.L., X.-W.D., C.M.L., M.F.T., Y.Y., Z.H., and S.Y.H. contributed new reagents/analytic tools;D.-L.Y., J.Y., C.-S.M., Y.Y., Z.H., and S.Y.H. analyzed data; and D.-L.Y., J.Y., C.-S.M., Y.Y., Z.H.,and S.Y.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession nos. GSE29577and GSM732294–GSM732299).1D.-L.Y., J.Y., and C.-S.M. contributed equally to this work.2Present address: Institute for Sustainable and Renewable Resources, Institute for Ad-vanced Learning and Research, Danville, VA 24540.

3To whom correspondence may be addressed. E-mail: yuy3@psu.edu, zhhe@sibs.ac.cn, orhes@msu.edu.

See Author Summary on page 7152 (volume 109, number 19).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1201616109/-/DCSupplemental.

E1192–E1200 | PNAS | Published online April 23, 2012 www.pnas.org/cgi/doi/10.1073/pnas.1201616109

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elongation. Most recently, it has been shown that DELLArepressors promote JA signaling through physically interactingwith JAZ1 (23), suggesting a mechanism for GA-mediateddown-regulation of JA defense responses. However, it remainsunknown how JA could inhibit plant growth. In this study,through analysis of rice and Arabidopsis, we have elucidateda molecular cascade by which JA antagonizes GA signaling thatexplains how monocot and dicot plants prioritize JA defenseover growth.

ResultsKnockdown of Rice COI1 Genes Decreases JA Response. As a modelmonocot, rice (Oryza sativa L.) has been commonly used to studyhormone signaling as well as defense responses in cereal crops. Anumber of studies have been conducted to dissect JA signalingand function in defense response and developmental process inrice (24–27). However, the role of COI1 in mediating JA signalperception is still unclear in rice. The rice genome contains twoclosely related COI1 genes, OsCOI1a (Os01g0853400) andOsCOI1b (Os05g0449500), which share 83% and 82% sequenceidentity at the DNA and protein levels, respectively. To de-termine the function of COI1 in rice, a double-strand RNAiconstruct containing the conserved sequence of OsCOI1a andOsCOI1b (Fig. S1A) was introduced into the model varietyNipponbare. More than 20 independent RNAi lines were pro-duced (Fig. S1B). The transcript levels of both OsCOI1a andOsCOI1b were significantly reduced in these RNAi lines asdetected by RNA blot and quantitative RT-PCR (qRT-PCR)analyses (Fig. 1 A and B), indicating that OsCOI1 expression waseffectively knocked down by RNAi.

JA sensitivity was investigated in two stable RNAi lines, coi1-13 and coi1-18, that carry a single copy of the transgene. In thisstudy, we used methyl JA (MeJA), which is converted to JA andthen to bioactive jasmonoyl-isoleucine in plants (3), to treat riceand Arabidopsis. As expected, the transgenic lines were much lesssensitive to MeJA than the WT plants in the growth-inhibitionassay (Fig. 1 C and D). Furthermore, JA-responsive genes suchas OsVSP and OsMPK7 exhibited reduced expression in theOsCOI1-RNAi plants (Fig. 1E). These results demonstrate thatOsCOI1 is required for JA signaling in rice.

OsCOI1-RNAi Plants Display Phenotypes Similar to Those of GAOverproduction. Intriguingly, when grown in the greenhouse orthe paddy field, the coi1-13 and coi1-18 plants consistently showedincreased plant height in comparison with the WT plants, a phe-notype that mainly resulted from elongated internodes (Fig. 2 A–C). This elongated phenotype of the OsCOI1-RNAi plants issimilar to that of the rice eui1 mutants, which contain a loss-of-function mutation in the P450 monooxygenase CYP714D pro-tein that catalyzes the 16α,17-epoxidation reaction of GA de-activation (28, 29). Another similarity between the rice coi1 linesand the eui1 mutants was that they produce longer grains thanthe WT plants (Fig. S2). Plant growth is controlled by cell di-vision and cell elongation. Cell length in the uppermost in-ternode of the coi1-18 plants was found to be significantlyincreased in comparison with that of the WT plants (Fig. 2 D andE), indicating that increased plant height is mainly caused bycell elongation instead of cell division, a GA-related feature(30). Whole transcriptomic analysis of coi1-13 plants revealedthat basal expression of several GA-related genes, includingGA2ox, GA20ox, and OsWRKY71, was altered in the OsCOI1-RNAi rice (Fig. S1C).GA signaling regulates diverse biological processes, including

α-amylase release, during seed germination in rice (31). We ex-amined the effect of MeJA on the GA induction of α-amylaseactivity in embryoless seeds, and found that coincubation withMeJA strongly suppressed the GA induction of α-amylase (Fig.S3A). Consequently, the seed germination rate was significantlydecreased with MeJA treatment (Fig. S3B). In contrast, theseeds of coi1-13 and coi1-18 had significantly higher levels ofα-amylase activity and germinated at a much quicker rate thanthose of the WT (Fig. S3 C and D). These results indicated thatthe modulation of GA signaling by JA occurs not only in plantgrowth but also during seed germination. Taken together, theseresults suggest that interruption of JA signaling in the coi1mutants augments the GA signal pathway in rice.

OsCOI1-RNAi Plants Are Hypersensitive to GA and Hyposensitive toGA Biosynthesis Inhibitor. To further confirm the alteration of GAsignaling in the OsCOI1-RNAi rice plants, the growth of riceseedlings was examined in semisolid one-half Murashige–Skoog(MS) medium supplemented with different concentrations ofGA3. The coi1-18 plants exhibited more sensitivity to exogenousapplication of GA3 in comparison with the WT plants (Fig. S4A).Consistent with their increased GA sensitivity, the coi1-18 plantsexhibited reduced sensitivity to the GA biosynthesis inhibitoruniconazole compared with the WT plants (Fig. S4B). Therefore,the OsCOI1-RNAi plants were hypersensitive to exogenous GAand hyposensitive to GA biosynthesis inhibitor.To examine the effect of the OsCOI1 silencing on the en-

dogenous GA levels, the bioactive GAs, GA1 and GA4, weremeasured in the RNAi and WT plants. In contrast to the eui1mutants, which accumulate extremely high levels (30- to 100-fold) of GA1 and GA4 (28), the coi1-18 plants accumulated onlyslightly higher levels of GA4 (3.8 fold) than the WT plants in theelongating uppermost internode (Table S1). The levels of GA1were similar in coi1-18 and WT plants. The modest change in theGA4 level may be correlated to the differential expression of

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Fig. 1. Generation of OsCOI1 RNAi transgenic rice with reduced JA sensi-tivity. (A) Suppression of OsCOI1a expression in transgenic RNAi lines asshown by RNA blot analysis. A fragment of 825 to 1,244 nt of OsCOI1 wasused as the probe, and 25S rRNA was used as the loading control. (B) Sup-pression of OsCOI1b expression in transgenic RNAi lines as shown by qRT-PCR. (C and D) The representative RNAi lines coi1-13 and coi1-18 show re-duced sensitivity to MeJA treatment. Seedlings were grown in one-half MSmedium with 0.6% agar supplemented with or without 20 μM MeJA. Pic-tures were taken at day 7 and shoot length was measured at day 12. Datashown in D are the means from 12 plants. Error bars represent SD. Asterisksindicate significant difference between WT and coi1 mutants based onStudent’s t test (P < 0.01). (E) Reduced expression of JA-response genesOsMPK7 and OsVSP in coi1-13 and -18 plants revealed by qRT-PCR. Datashown in C and E are the means from two independent experiments. Errorbars represent SD. Asterisks indicate the significant difference between WTand coi1 mutants based on Student’s t test (P < 0.01).

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several GA metabolism genes in OsCOI1-RNAi plants (Fig.S1C). However, overall the defect in JA signaling does not ap-pear to dramatically affect bioactive GA biosynthesis/accumu-lation in rice, even though OsCOI1-RNAi plants exhibited eui1-like growth phenotypes. Consistent with this observation, nosignificant difference in the transcript levels of EUI1 was foundbetween coi1-18 and the WT (Fig. S5). Taken together, theseresults strongly suggest that the increased plant height and cel-lular elongation of the OsCOI1-RNAi plants is mainly a result ofthe hypersensitivity to GA.

Elongation of OsCOI1-RNAi Plants Is Inhibited by Attenuating GASignaling. EUI1 overexpression resulted in a series of GA-de-ficient phenotypes, with drastic reduction of the bioactive GAs(28) and accumulation or stabilization of the DELLA proteinSLR1 (32). We crossed the EUI1-overexpression plants (Eui1-OX)to the coi1-18 plant (Fig. 3D). The homozygous Eui1-OX/coi1-18plants showed greatly reduced plant height (Fig. 3 A–C) and re-duced cell size in the uppermost internode (Fig. 3 E and F), similarto Eui1-OX plants. In addition, the longer grain phenotype of thecoi1-18 plants was also reverted to that of the WT (Fig. 3 G andH). We also crossed coi1-18 with the GA receptor gid1-1 mutant.Again, the gid1-1/coi1-18 double mutant exhibited a dwarf phe-notype like gid1-1 (Fig. S6). This result demonstrated that the GAreceptor gene GID1 is required for the function of OsCOI1 in theGA pathway. These results suggest that the OsCOI1-RNAi mor-phology is dependent on the GA signaling pathway.

JA Antagonizes GA Signaling Pathway by Delaying GA-Induced SLR1Degradation. We next wanted to determine the level of SLR1,a rice DELLA protein that functions as a key repressor of theGA signaling pathway (33). However, it was difficult to detectSLR1 in the coi1-18 or the WT plant because of the low basallevel of SLR1. Instead, we found that SLR1 accumulated in theEui1-OX/coi1-18 plant at a level comparable to the Eui1-OXplant (Fig. 4A). Therefore, the degradation dynamics of SLR1protein was examined in Eui1-OX/coi1-18 in the presence of

exogenous GA3. When transferred into the medium with 100 μMGA3 for 30 min, SLR1 was significantly degraded in the Eui1-OX/coi1-18 plants, whereas it was degraded only slightly evenafter 2 to 3 h with GA treatment in the Eui1-OX plants (Fig. 4A).This result suggested that JA signaling antagonizes GA-mediatedreduction of the DELLA protein.Having shown that turning down the JA pathway could in-

crease the GA signaling output, we next examined the possibilitythat turning on JA signaling might antagonize the GA signalingpathway. Indeed, whereas growing WT rice seedlings in thepresence of 10 μM GA3 leads to elongation of the second leafsheath by approximately 120%, addition of MeJA greatly re-duced the GA-triggered elongation in a dose-dependent manner(Fig. 4 B and C). Furthermore, in the presence of MeJA, GA-induced SLR1 degradation was significantly inhibited as long as6 h after treatment (Fig. 4D). Consistent with this observation,seedling growth was inhibited by MeJA in a dose-dependentmanner, with decreased shoot length (Fig. 4 E and F). In addi-tion, the SLR1 protein accumulated in the plants grown in themedium supplemented with MeJA (Fig. 4G), whereas no changewas observed in the SLR1 transcript level (Fig. S7). Finally, thegrowth inhibition effect of MeJA was significantly suppressed inthe SLR1 loss-of-function mutant slr1 in comparison with theWT (Fig. 5), further supporting that JA-mediated growth in-hibition is in part dependent on the DELLA repressor. Theseresults collectively demonstrated that JA represses rice growththrough antagonizing GA signaling at least partly via affectingthe level of the DELLA protein SLR1.

Arabidopsis coi1 Mutant Also Exhibits GA-Related Phenotypes. Thesignificant GA hypersensitivity phenotypes of the OsCOI1 RNAilines was somewhat unexpected because such phenotypes werenot previously reported for the Arabidopsis coi1 mutants (6, 34).We therefore looked for GA-related phenotypes in the Arabi-dopsis coi1 mutant plants. We found that Arabidopsis coi1 plantshave several robust phenotypes that resemble GA hypersensi-tivity, including longer hypocotyls and petioles under low-in-

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Fig. 2. Morphological phenotypes of coi1-18 plants. (A) Images of WT (Nipponbare) and coi1-18 plants show plant heights and internode lengths. (B)Quantification of heights of the WT, coi1-13, and coi1-18 plants. More than 30 plants of each line were analyzed. The difference between the control andtransgenic plants is significant (**P < 0.001, Student’s t test). (C) Lengths of individual internodes in the WT and coi1-18 plants. Each internode of coi1-18 waslonger than the counterpart of Nipponbare (*P < 0.01 and **P < 0.001, Student’s t test). (D) Microscopic sections of the elongating zone of the uppermostinternodes from Nipponbare and coi1-18 grown in the isolated paddy field. (Scale bar, 40 μm.) (E) Cell lengths at the base of the elongating zone of theuppermost internodes in the WT and coi1-18 plants. The cell of coi1-18 is much longer than that of Nipponbare (**P < 0.001, Student’s t test).

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tensity light condition and early flowering (Fig. 6). Moreover,transgenic overexpression of JAZ repressors, which mimics theeffect of coi1 mutations, was found to phenocopy the coi1 mu-tant. Among eight AtJAZ genes (AtJAZ1, 3, 4, 5, 6, 9, 10, and 11)we were able to overexpress, AtJAZ1, 3, 4, 9, 10, and 11 producedthe early flowering phenotype, but, interestingly, AtJAZ5 and 6overexpression plants did not (Fig. S8). We also checked AtJAZ9overexpression plants for GA-mediated germination responseand found that they were more resistant to the GA biosynthesisinhibitor paclobutrazol (Fig. S9), which is a GA-hypersensitivityphenotype.Next, we investigated whether, like in rice, JA could antago-

nize GA signaling by affecting the level of DELLA proteins inArabidopsis. A well characterized DELLA protein, RGA, wasmonitored in these experiments. Consistent with what was ob-served in rice, when Arabidopsis seedlings were continuouslytreated with JA, the RGA protein level increased, whereas theRGA transcript level did not change (Fig. 7). As internal controls,JA induced degradation of JAZ9 and expression of a known JA-

responsive gene, AOS (Fig. 7). Taken together, these resultscollectively show that disruption of JA perception and signalingaffects GA phenotypes in Arabidopsis and that JA negativelyregulates GA responses through modulating the level of DELLArepressors in rice and Arabidopsis.

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Fig. 4. Levels of the rice DELLA protein SLR1 and the antagonistic effect ofMeJA on GA-mediated plant growth. (A) The GA-mediated degradation ofthe DELLA protein SLR1 was promoted in the coi1-18 plants. The 10-d-oldseedlings grown on one-half MS plates with 0.6% agar were transferred toliquid one-half MS medium with 100 μM GA3, and the SLR1 protein wasdetected with an SLR1 antibody at the indicated time points. (B and C) MeJAinhibited GA-induced shoot elongation in WT plants: 1, control plant; and 2to 5, representative plants treated with GA3 and MeJA in the same order asin Fig. 4C. The relative growth is indicated by the length of second leafsheath after being treated with 10 μM GA3 and various concentrations ofMeJA. (D) MeJA delayed SLR1 degradation induced by GA3. The concen-tration of GA3 and MeJA used were 10 μM and 100 μM, respectively. (E andF) MeJA inhibited WT rice seedling growth and second sheath elongation ina dose-dependent pattern. Asterisks indicate significant difference betweenmock and treatments (P < 0.01, Student’s t test). (G) MeJA treatment pro-moted the accumulation of SLR1 in a dose-dependent manner in WT plants.The rice plants were grown on one-half MS plates with 0.6% agar supple-mented with MeJA (final concentration indicated on Top).

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JAZ Repressors Directly Interfere with DELLA–PIF Interaction.DELLA proteins have been shown to interact and repressgrowth-promoting transcription factors, such as PIFs in Arabi-dopsis (18, 19). Interestingly, the DELLA proteins were recentlyfound to also interact with AtJAZ1 in Arabidopsis (23). By usingmultiple methods, we independently observed multiple JAZ–DELLA interactions in plant or yeast, and found that, in the caseof the JAZ9–GAI interaction, the N terminus of JAZ9 and theGRAS domain of GAI are important for interaction in yeast*(Fig. 8 A and B and Fig. S10). Although Hou et al. focused theirstudy on how GA antagonizes JA signaling through the AtJAZ1–DELLA interaction, we noticed a striking correlation betweenthe ability of AtJAZ overexpression to confer early flowering(Fig. S8) and physical interaction with DELLA proteins:AtJAZ1, 3, 4, 9, 10, and 11, but not AtJAZ5 and 6, interactedwith DELLA proteins and produced the early flowering pheno-type (Fig. S10). We therefore investigated the intriguing possi-bility that AtJAZ repressors may impede the DELLA–PIFinteraction. We first confirmed the interaction between theGRAS domain of RGA and PIF3 in a yeast two-hybrid assay(Fig. 8C). We found that expression of AtJAZ9 inhibited theRGA–PIF3 interaction in yeast three-hybrid assays (Fig. 8C),without affecting the protein levels of RGA and PIF3 (Fig. 8D).The effect of JAZ9 on the RGA-PIF3 interaction could also beobserved in plant cells by using Nicotiana tabacum-based tran-sient expression assays. Again, RGA interacted with PIF3 in this

system; however, coexpression with AtJAZ9 could effectivelyprevent RGA–PIF3 interaction (Fig. 8E).Our results suggest that JAZ-mediated interference with the

DELLA–PIF interaction is a key mechanism that modulatesplant growth. To obtain genetic evidence for or against thispossibility, we analyzed the responses of pif mutants and PIF3overexpression plants to JA treatment. We found that the pifquadruple mutant (pifq) grew more slowly compared with WTplants, and were no longer able to respond to JA-mediated in-hibition of hypocotyl growth (Fig. 9 A and B). This result sug-gests that PIFs are likely the main, if not the only, growth-promoting transcription factors that are targeted by JA-inducedgrowth inhibition. More interestingly, overexpression of PIF3alone was sufficient to partially overcome JA-induced inhibitionof hypocotyl growth (Fig. 9 A and B). Our results contrast withthose from a recent report that showed that PIF4 transgenicoverexpression plants exhibited enhanced JA-induced growthinhibition (23). Also, although the della quadruple mutant (del-laq) showed only a slightly lower sensitivity to JA-mediated in-hibition of hypocotyl growth in the study by Hou et al. (23),under our experimental conditions, the della quintuple mutant(gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1) was almost completely insen-sitive to JA-induced hypocotyl inhibition (Fig. S11). Finally, wefound that expression of two examined DELLA/PIF-regulatedgenes—expansin 10 (EXP10, At1G26770) and xyloglucan:xylo-glucosyl transferase 33 (XTH33, At1G10550) (35–37)—was al-tered in a predicted manner upon JA treatment: JA up-regulatesthe expression of EXP10, which is down-regulated by PIFs,whereas JA down-regulated XTH33, which is up-regulated byPIFs (Fig. 9 C and D). Taken together, our results stronglysuggest that JAZ-mediated interference with the DELLA–PIFinteraction is a critical part of a mechanism by which JAantagonizes GA signaling in modulating growth.

DiscussionThe growth–defense conflict is a widely known phenomenon inplants, although the underlying molecular mechanism is not well

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Fig. 6. GA-associated phenotypes of the Arabidopsis coi1 mutant and JAZ9overexpression plants. Image (A) and quantification (B) of petioles of coi1-30and JAZ9 overexpression plants. The plants were grown in a long-daygrowth chamber (16 h 120 μmol m−2·s−1 light/8 h dark, 22 °C/18 °C). The thirdtrue leaves of 21-d-old plants were imaged, and their petiole lengths weremeasured. Image of 28-d-old plants (C) and quantification (D) of earlyflowering phenotypes of JAZ9 overexpression lines and coi1-30 plants. Theplants were grown under the same conditions as in A. Image (E) andquantification (F) of hypocotyls of coi1-30 and JAZ9 overexpression plantswhen grown under 10 μmol m−2·s−1 continuous white light at 22 °C for 6 d.(Scale bar, 5 mm.) Data shown in B, D, and F are the means from 12 plants.Error bars represent SD. Letters on columns indicate significant differences(P < 0.05, Tukey–Kramer multiple comparison test).

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Fig. 7. MeJA increases the level of the Arabidopsis DELLA protein RGA.Seedlings were grown under 10 μmol m−2·s−1 continuous white light at 22 °C.(A) Increased accumulation of the RGA protein in Arabidopsis seedlingstreated with different concentrations of MeJA for 6 d. RGA and JAZ9 (pos-itive control) were detected by anti-GFP and anti-HA antibody, respectively.(B) MeJA treatment does not affect the RGA transcript level in Arabidopsis.Arabidopsis seedlings were treated with 0.1% ethanol (mock) or 100 μMMeJA for 6 d. Total RNA was purified and used for qRT-PCR analysis. Datashown are the means of three biological replicates. Error bars represent SD.JA-inducible expression of AOS was used as a positive control for JA treat-ment. Asterisks indicate significant difference between mock and MeJAtreatment (P < 0.01, Student’s t test).

*Yao J, Withers J, He SY, Plant Biology 2011, August 6–10, 2011, Minneapolis, MN,abstr P16037.

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characterized. JA is an important plant hormone that playsa prominent role in plant defense against diverse pathogens andherbivores (3, 38). Despite rapid progress on dissecting the JAsignaling pathway in recent years, a mechanistic explanation forhow plants effectively balance growth and defense in response tothe activation of JA signaling has remained elusive. In this study,we show that modulation of the level of DELLA repressors andinterference with DELLA interaction with growth-promotingPIF transcription factors are two key mechanisms underlyingJA-mediated growth inhibition in monocot rice and dicot Ara-bidopsis, illustrating a potentially widely conserved strategy bywhich angiosperm plants coordinate a major form of growth/defense tradeoff. Future research shall address whether the twomechanisms function independently of each other and how JAsignaling modulates the level of DELLA proteins.The DELLA proteins were first identified as key repressors of

the GA pathway (39), and were subsequently shown to impactother hormone pathways such as auxin, abscisic acid, and eth-ylene (36, 40–42); plant photomorphogenesis (18, 19, 43); andplant survival under abiotic stress (44–46). Our results furthersupport the notion that DELLA proteins act as key regulators/switches in integrating hormone and environmental signals andin fine-tuning plant growth and stress responses (47), which arecritical for plant survival under harsh conditions (44, 46). Wehave provided clear evidence that JA treatment increases SLR1levels in rice and RGA in Arabidopsis, which are predicted toresult in the repression of plant growth. Conversely, we foundthat JAZ9 could effectively interrupt RGA–PIF3 interaction,suggesting that, in the absence of JA signaling, some DELLArepressors could be titrated out by JAZ proteins, which wouldallow more PIF transcription factors to activate growth pro-grams. This mechanism could explain the GA-hypersensitivityphenotypes observed in the coi1 mutants of rice and Arabidopsisand in transgenic Arabidopsis plants overexpressing those JAZproteins (e.g., AtJAZ1, 3, 4, 9, 10, and 11) that interact withDELLA proteins. It can also explain why not only the dellaquintuple mutant was largely insensitive to JA-induced growthinhibition, as expected, but also why overexpression of PIF3 couldpartially counter JA inhibition of growth (Fig. 9A). In short, wehave provided experimental evidence for a signaling cascade,involving the COI1–JAZ–DELLA–PIF signaling module, thatunderlies the growth inhibition during JA defense activation.In this study, we noticed interesting differences between rice,

a domesticated monocot crop, and Arabidopsis, a wild dicot, inthat prominent GA phenotypes of coi1 mutants are displayedunder different conditions for rice and Arabidopsis. WhereasOsCOI1-RNAi plants exhibit exaggerated stem elongation andother GA-related phenotypes under strong light conditions inthe greenhouse and in the field, Atcoi1 plants show most obviousGA phenotypes under dim light conditions (10 μmol m−2·s−1

continuous white light; Materials and Methods), but not underother growth conditions previously reported (6, 34). Therefore,although the core JA and GA pathways are likely conserved inmonocot and dicot plants, divergence in JA and GA signalingand/or the crosstalk between JA and GA signaling in dicot andmonocot species might exist. Recently, Robson et al. (48) foundthat the coi1 mutant flowered earlier and developed longer

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interaction between RGA and PIF3, is greatly reduced [indicated by reducedgrowth on medium lacking histidine (-His)] in the presence of JAZ9 [inducedin medium without methionine (-Met)]. (D) Western blot shows that allproteins analyzed in the Y3H assay (C) were expressed as expected. (E) 3xHA-JAZ9 interferes with the interaction between 9xMyc-RGA and 3xFLAG-PIF3when transiently expressed in N. tabacum leaves. Protein extracts wereimmunoprecipitated with an anti-Myc antibody and analyzed by Westernblot with anti-FLAG, anti-HA, or anti-Myc antibody.

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hypocotyls under low red/far-red light conditions than the WT. Itis possible that these phenotypes could also be related to GAphenotypes studied here because light and GA signals integrateto regulate Arabidopsis growth (18, 19, 44), with DELLA pro-teins functioning in plant photomorphogenesis (43). We there-fore propose that DELLA-mediated integration of JA, GA, andlight signaling may give rise to a fundamental framework andneeded flexibility in JA-induced growth–defense tradeoff in ad-aptation to and/or reflecting extraordinarily diverse growthhabitats and domestication histories of angiosperm plants.

Materials and MethodsPlant Materials and Growth Condition. Rice plants (cv. Nipponbare) weregrown in a greenhouse or in the isolated paddy field. All Arabidopsis plantsdescribed here were derived from Col-0 except for the della mutant, whichis in Landsberg erecta (Ler) background. The jaz9-1, dellaq (gai-t6/rga-t2/rgl1-1/rgl2-1/rgl3-1), and pifq (pif1-1/pif3-3/pif4-2/pif5-3) mutants, as well asthe PRGA::GFP-RGA and PIF3OE transgenic lines, have been previously de-scribed (9, 18, 35, 49, 50). The jaz9-3 (SM_3_34031; Fig. S12) and coi1-30(SALK_035548; Fig. S13) mutants were characterized in the present study.All Arabidopsis seeds were ordered from the Arabidopsis BiologicalResource Center.

Arabidopsis seeds were stratified for 3 d at 4 °C before planting. Surface-sterilized seeds were sown on MS medium containing 0.8% agar and 5 mMMes (pH 5.8), and placed in a growth chamber with 10 μmol m−2·s−1 con-tinuous cool-white fluorescent light at 22 °C or in a long-day growthchamber with a 16-h day (120 μmol m−2·s−1 cool-white fluorescent light,22 °C) and 8-h night (18 °C) cycle. The soil-grown plants were placed in thelong-day growth chamber.

Transgenic Expression. For generation of OsCOI1-RNAi transgenic rice, twofragments of the OsCOI1 genes were amplified by using the primer pairsOsCOI1-F1/R1 and OsCOI1-F2/R2 (Dataset S1), respectively. The fragmentswere inversely inserted into the pCAMBIA1300S that contained a double 35Spromoter and a terminator. The resulting OSCOI1 RNAi construct was in-troduced into the model variety rice Nipponbare (Oryza sativa L. ssp. ja-ponica) by using Agrobacterium-mediated transformation. IndependentRNAi rice lines were analyzed and confirmed by Southern and Northern blotanalyses, as well as qRT-PCR assays. All transgenic plants were grown ina greenhouse or in the isolated paddy field for measurement of plant heightand other morphological traits.

For production of transgenic Arabidopsis lines expressing P35S::3×HA-AtJAZ9-8×His, the coding sequence of AtJAZ9, excluding the stop codon,

was amplified by PCR by using PfuUltra II DNA polymerase (Agilent Tech-nologies) and Arabidopsis cDNA, which was obtained from a 28-d-old Col-0 plant by using the AtJAZ9-F and -R primer set (Dataset S1). The PCRproduct was first cloned into vector pGEM-T easy (Promega), and thenmoved into a binary vector pJYP003 (SI Materials and Methods) to createa 3×HA-AtJAZ9-8×His fusion construct. Col-0 plants were transformed byAgrobacterium-mediated transformation. Primary transformants were se-lected based on BASTA resistance, and the JAZ9 expression level was de-termined by Western blotting. Transgenic lines with a 3:1 (resistant:sensitive)segregation ratio for BASTA resistance were selected, and several homozy-gous lines were identified in the T3 generation.

The Gateway entry clones containing GAI, RGA, or PIF3 were identifiedfrom the REGIA Arabidopsis transcription factor library (51). Inserts weretransferred into pJYP006 or pJYP012 by LR recombination (Life Technolo-gies) to create P35S::9×Myc-GAI, P35S::9×Myc-RGA, and P35S:3×FLAG-PIF3,which were used for transient expression in tobacco leaves as previouslydescribed (52).

Hormone Treatment and Growth Assay. For rice, the seeds were sterilized andincubated on one-half MS medium with 0.6% agar and supplemented withdifferent concentrations of GA3 and MeJA. Seedling (i.e., shoot) growth andthe lengths of the second sheath were measured 12 d after treatment. ForWestern blotting, 10-d-old seedlings grown in one-half MS medium with0.6% agar were transferred to liquid one-half MS medium supplementedwith 100 μM GA3 with or without 100 μM MeJA. Samples were harvested atdifferent time points and frozen at −80 °C for RNA and protein extraction.

Arabidopsis seeds used for growth assays were harvested on the same dayfrom plants grown side by side. Seedlings were grown on MS plates for 4 dbefore being transferred onto soil. Seedlings of homozygous coi1-30 plantswere selected on MS plates containing 10 μM MeJA (Sigma-Aldrich). Plantswere kept in a long-day growth chamber unless indicated otherwise. Flow-ering time was determined when floral buds became visible at the center ofrosette. Petiole lengths of the third true leaves were measured on day 21. Atleast 16 plants of each line were assessed.

Arabidopsis seedlings were also grown on plates with or without 10 μMMeJA under continuous light for 6 d. Seedlings were then placed on a newplate and scanned at a resolution of 600 dpi. The hypocotyl length wasmeasured by using ImageJ software (National Institutes of Health).

Endogenous GA Assay in Rice. The elongating uppermost internodes of thetransgenic andWT plants were harvested and lyophilized at −20 °C. GAs wereextracted, and GA1 and GA4 were assayed by LC-MS with internal standardsas described previously (53).

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2·s−1 continuous white light at 22 °C for 6 d. Image (A) and quantification (B) of the effect of MeJA on Arabidopsis hypocotyl elongation. The hypocotyllengths were measured and the inhibition of hypocotyl growth was calculated as (1 − treated / untreated) × 100%. Data shown are the means from 16seedlings. Error bars represent SD. Letters on columns indicate significant differences (P < 0.05, Tukey–Kramer multiple comparison test). (C and D) MeJA hasantagonizing effects on the expression of XTH33 (PIF-up-regulated) and EXP10 (down-regulated) genes in Arabidopsis. Total RNAs were purified and used forqRT-PCR analysis. Data shown are the means of three biological replicates. Error bars represent SD. Asterisks indicate significant difference between mock andMeJA treatment (P < 0.05, Student’s t test).

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RNA Blot and Transcript Analysis. Total RNA was isolated from rice leaf tissuesby using TRIzol reagent according to the manufacturer’s protocol (Invi-trogen). RNA gel blotting was performed by using standard protocol withPerfectHyb buffer (Sigma), and relative gene expression was quantified byusing a PhosphorImager (Amersham Biosciences). For qRT-PCR, total RNAwas first treated with DNase I and the first-strand cDNA was then synthe-sized by using the oligo dT primer and SuperScript II reverse transcriptase(Invitrogen). Rice ubiquitin 1 gene (UBQ1; Os06g0681400) was used as aninternal control to normalize samples. Quantitative PCR was performed onthe Mx3000P real-time PCR system (Agilent Technologies) with a QuantiTectSYBR Green PCR kit (Qiagen). Semiquantitative RT-PCR was conducted by us-ing the SuperScript III First-Strand Synthesis System (Invitrogen). The primersused to detect the transcripts of the target genes are listed in Dataset S1.

For analysis of Arabidopsis transcripts, total RNA was extracted by usingan Ambion ToTALLY RNA Total RNA Isolation Kit (Life Technologies)according to the manufacturer’s manual. After DNase I (Roche) treatment,RNA was further purified by using an RNeasy Mini Kit (Qiagen). First-strandcDNA was synthesized by using M-MLV reverse transcriptase (Invitrogen)and oligo(dT) as primers. actin2 (AT3G18780), cap-binding protein 20(CBP20; AT5G44200), protein phosphatase 2A subunit A3 (PP2AA3;AT1G13320), and ubiquitin-conjugating enzyme 21 (UBC21; AT5G25760) (54)were used as internal controls to normalize target gene expression byGeNORM method (55). Quantitative PCR was performed on an ABI7500 FastReal-time PCR System with Fast SYBR Master Mix (Applied Biosystems)according to the manufacturer’s protocol. The primers used to detect spe-cific transcripts are listed on Dataset S1.

Protein Extraction, Quantification, and Immunoblots. Total proteins wereextracted using plant protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1 mM DTT) with 1%protease inhibitor for plant cell and tissue extract (Sigma-Aldrich) and 100 μMMG132 (Cayman Chemicals). Protein content was quantified by using theProtein DC assay kit (Bio-Rad). Equal amounts of protein were subjected toSDS/PAGE followed by Western blotting analysis. Immunodetection of GFP-RGA, HA-JAZ9, RGA, Myc-RGA, Myc-GAI, FLAG-PIF3, and SLR1were per-formed by using anti-GFP antisera (Abcam), anti-HA antibody (Roche Ap-plied Science), anti-RGA antisera (49), anti-Myc antisera (Abcam), anti-FLAGantibody (Sigma-Aldrich), and anti-SLR1 (33), respectively. CorrespondingHRP conjugated secondary antibodies and SuperSignal West Pico Chemilu-minescent Substrate (Thermo Fisher Scientific) were used for detection.

Coimmunoprecipitation Assay. Total proteins were extracted from Arabi-dopsis seedlings or tobacco leaves by using lysis buffer (50 mM Tris-HCl, pH7.5, 75 mM NaCl, 0.2% Triton X-100, 5 mM EDTA, 5 mM EGTA, 1 mM DTT,1% Sigma Protease inhibitor mixture,100 μM MG132, 10 mM NaF, and 2 mMNa3VO4). The immunocomplexes were captured by anti-HA or anti-Mycagarose beads (Sigma-Aldrich), washed, and released by 2× SDS samplingbuffer, and were then subjected to SDS/PAGE followed by further Westernblotting analysis.

Rice Microarray Assay.Whole transcriptomic analysis was performed with theAffymetrix GeneChip Rice Genome Array, representing 51,279 transcriptswith three biological replicates. Raw data were analyzed with AffymetrixGeneChip Operating Software (GeneSpring, version 11.0) using Affymetrixdefault analysis settings and global scaling as normalization method. The GA,JA, and defense-related gene was analyzed by MAPMAN. The microarray

data has been deposited into the National Center for Biotechnology In-formation Gene Expression Omnibus database (accession nos. GSE29577 andGSM732294–GSM732299).

Assays for α-Amylase Activity. The release of α-amylase was assayed as de-scribed by Zhang et al. (29). The sterilized embryoless half seeds were in-cubated on agar plates containing 0.2% starch with 1 μM GA3 or 1 μM GA3

and 50 μM MeJA for 3 d at 28 °C in darkness. These plates were exposed toiodine vapor for a few minutes. The reaction between starch and iodineturned the agar plates a blue-purple color. The agar around half seeds withα-amylase activity remained colorless because of hydrolysis of starch byα-amylase. The α-amylase protein was extracted from deembryonated halfseeds imbibed in 1.0 μM of GA3 solution in the dark at 28 °C for 2 d (56). Theα-amylase activity was assayed by quantifying reducing sugar released fromsubstrate starch (57).

Microscopic Observation. Rice internodes and sheaths were sampled for resinsectioning. The samples were first fixed in FAA [3.7% (vol/vol) formaldehyde,5% (vol/vol) acetic acid, 50% (vol/vol) ethanol], followed by dehydrationthrough a graded ethanol series. The samples were embedded in resin andpolymerized at 58 °C. Sections (8–10 μm) were examined under a microscope(DMLB; Leica) and documented by photography.

Yeast Three-Hybrid Assay. The coding sequence corresponding to the GRASdomain of RGA was amplified by PCR by using the RGAdN-F and -R primer set(Dataset S1) and cloned into pGBridge (SI Materials and Methods) to createa Gal4DB–Myc–RGAΔN construct. The AtJAZ9 was cloned into pET42a-3×FLAG (SI Materials and Methods) to create a 3×FLAG-JAZ9 cassette, whichwas further transferred into pBridge (Clontech) to create PMET::3×FLAG-JAZ9. The PMET::3×FLAG-JAZ9 insert was transferred into the Gal4DB–Myc–RGAΔN fusion plasmid to create the yeast three-hybrid (Y3H) bait vector.The AtPIF3was cloned into pDEST-GADT7 (58) by LR recombination to createthe prey vector. The bait and prey vectors were transformed into the yeaststrain AH109 (Clontech). Y3H assays were performed following the manu-facturer’s protocol (Clontech).

Statistic Analysis. Unless indicated otherwise, one-way ANOVA was per-formed for all data sets. Tukey–Kramer multiple comparison test was used tocompare the means of the treatments at an α level of 0.05.

ACKNOWLEDGMENTS. We thank Dr. M. Matsuoka (Nagoya University) forthe gid1-1 mutant and the antibody against SLR1, Dr. Q. Qian (China Na-tional Rice Research Institute) for the slender rice 1 mutant, Dr. J.-M. Lifor useful discussion on the research, Dr. P. Quail (University of California)for pifq mutant and PIF3 antibody, H. Zhang, R. K. Kalia, and J. Withers forcritical reading of the manuscript, and J.-Q. Li and X.-Y. Gao for microscopyassistance. This work was supported by Natural Science Foundation of ChinaGrants 90817102 and 30730064 (to Z.H.), National Key Basic Research andDevelopment Program Grant 2011CB100700 (to Z.H.), US Department ofAgriculture National Research Initiative Grant 2003-35319-17873 (to Y.Y.),National Science Foundation Plant Genome Research Program Grant DBI-0922747 (to Y.Y.), National Institutes of Health Grant R01AI068718 (to S.Y.H.for experiments with jasmonate signaling), and US Department of Energy(Chemical Sciences, Geosciences, and Biosciences Division, Office of BasicEnergy Sciences, Office of Science) Grant DE-FG02-91ER20021 (to S.Y.H. forexperiments with growth and GA signaling). S.Y.H. is a Howard HughesMedical Institute and Gordon and Betty Moore Foundation Investigator.

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